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Aedes aegypti Act4  is a paralog of the  Drosophila melanogaster  indirect flight muscle actin gene  Act88F .  Act88F  has been shown to be haploinsufficient for flight in both males and females (amorphic mutants are dominant). Whereas  Act88F  is expressed in indirect flight muscles of both males and females, expression of  Act4  is substantially female-specific. We therefore used CRISPR/Cas9 and homology directed repair to examine the phenotype of  Act4  mutants in two Culicine mosquitoes,  Aedes aegypti  and  Culex quinquefasciatus . A screen for dominant female-flightless mutants in  Cx .  quinquefasciatus  identified one such mutant associated with a six base pair deletion in the  CxAct4  coding region. A similar screen in  Ae .  aegypti  identified no dominant mutants. Disruption of the  AeAct4  gene by homology-dependent insertion of a fluorescent protein marker cassette gave a recessive female-flightless phenotype in  Ae .  aegypti . Reproducing the six-base deletion from  Cx .  quinquefasciatus  in  Ae .  aegypti  using oligo-directed mutagenesis generated dominant female-flightless mutants and identified additional dominant female-flightless mutants with other in-frame insertions or deletions. Our data indicate that loss of function mutations in the  AeAct4  gene are recessive but that short in-frame deletions produce dominant-negative versions of the AeAct4 protein that interfere with flight muscle function. This makes  Act4  an interesting candidate for genetic control methods, particularly population-suppression gene drives targeting female viability/fertility.

Targeting female flight for genetic control of mosquitoes